Infra-red spectroscopy system

ABSTRACT

A sample slide ( 100 ) for use in a spectrometer ( 501 ), wherein the sample slide comprises a plurality of sample-receiving portions ( 111 - 114 ) provided on a sample side ( 115 ) of the slide, and a plurality of beam-receiving portions ( 121 - 124 ) provided on a beam-receiving side ( 125 ) of the slide, each beam-receiving portion being arranged opposite a respective sample-receiving portion, and wherein each beam-receiving portion is configured to act as an internal reflection element (IRE). A device ( 300 ) for use with a spectrometer ( 501 ) comprises a stage ( 330 ) configured to receive a sample slide ( 100 ); and a moving mechanism ( 360 ) configured to move the sample slide relative to a sample-measuring location ( 320 ) of the device. Associated methods for preparing a sample and measuring a sample are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of, and claims priority to, U.S.patent application Ser. No. 16/496,053, filed Sep. 20, 2019, now U.S.Pat. No. 11,215,553, issued on Jan. 4, 2022, which is a 371 U.S.National Entry of patent application PCT/GB2018/050821, filed Mar. 28,2018, now expired, claiming priority to United Kingdom patentapplication 1714643.2, filed Sep. 12, 2017, now expired, and UnitedKingdom patent application 1705221.8, filed Mar. 31, 2017, now expired,the contents of which are incorporated herein in their entirety.

The present invention relates to apparatuses and methods for performingInfrared spectroscopy analysis, and in particular, though notexclusively, for performing ATR-FTIR spectroscopy analysis.

BACKGROUND OF THE INVENTION

Fourier Transform Infrared (FTIR) spectroscopy is a technique commonlyused in chemical sciences in order to identify discrete vibrations ofchemical bonds. This technique uses light in the mid-infrared (MIR)region (4000-400 cm⁻¹) that is, in the same frequency range as thefrequency range of chemical bond vibrations.

Biological molecules are known to actively vibrate in this range ofwavelengths, and thus FTIR spectroscopy lends itself to biologicalapplications. When a biological sample is irradiated with MIR light,some of this energy is absorbed by the sample. The absorption profile ofa given sample is representative of the chemical bonds present within asample, and can be used to characterise complex biological materials.

An example of a particular type of analysis using FTIR spectroscopy isin the investigation of proliferative disorders, such as cancer, whichare caused by uncontrolled and unregulated cellular proliferation andcan, in some cases, lead to the formation of a tumour.

There are three principal sampling modes used in FTIR spectroscopy:transmission, transflection, and attenuated total reflection (ATR).

In the “transmission” mode, MIR light is passed, or transmitted,directly through a given sample that has been deposited on an IRtransparent substrate (such as CaF₂ or BaF₂). As this mode is reliantupon the IR beam passing through the sample, there are constraints tomaximum sample thickness and water content.

In the “transflection” mode, a sample is deposited on an IR reflectiveslide (such as low-E or metal coated). MIR light is passed through thesample and it is then reflected back towards the detector. As the beamis effectively passed through the sample twice, the sample thickness hasa direct effect on pathlength and therefore signal strength. This alsoallows further absorption of water, if at all present in the sample.There are some known concerns in the field regarding this form ofsampling due to undetermined interaction of light with the reflectivesurface of the substrates.

“Attenuated Total Reflection” (ATR) employs an internal reflectiveelement (IRE) through which the IR beam is passed. The sample isdeposited directly onto the IRE, and maintained in close contact withit. These IREs can be made from a number of different materials,including diamond, germanium, zinc selenide or silicon. Each materialdiffers slightly in its refractive properties. When IR light is passedthrough an IRE above a defined angle, described as the critical angle,the light is internally reflected through this medium. When the beammeets the IRE and sample interface, this results in the production of anevanescent wave which penetrates into the sample. The depth of thispenetration is dependent upon the wavelength of light, the refractiveindices of the IRE and the sample, as well as the angle of incidence:however, is generally in the region between 0.5-2 μm. The beam is thenreflected by the IRE towards a detector.

One benefit of ATR-FTIR is the reduced influence of water absorbance onthe IR spectrum, allowing the interrogation of water-containing samples.This is particularly important to biological samples which willintrinsically contain water. Although water molecules still absorb inthis sampling mode, the penetration depth of the evanescent wave is muchsmaller than the pathlength of transmission and transflection FTIRspectroscopy. Therefore, much less water is being sampled, allowing theunderlying sample absorbance to still be monitored.

This technique has therefore lent itself well to the analysis ofbiological samples, particularly biofluids. These are known to beinformation rich and have been shown to be suitable for the detection ofdisease in a patient population. It has been shown that this techniqueis capable of diagnosing brain tumours at a range of severities usingblood serum from a cohort of 433 patients (Hands et al., 2016; Hands etal. 2014).

Recently a method of diagnosing brain cancer by performing AttenuatedTotal Reflection—Fourier Transform Infrared (ATR-FTIR) spectroscopicanalysis of blood samples has been described in WO 2014/076480). Incontrast to conventional ATR-IR (where a sample is placed on a substratethat is then brought into contact with the ATR crystal), the ATR crystalwas used as the substrate for the sample. This method provides a pointof care and non-destructive diagnostic test. However, it requirescareful preparation of the blood samples prior to carrying out aspectroscopic analysis, and thorough cleaning and drying of the ATRcrystal before the ATR crystal can be reused for analysis of anothersample, which is both time-consuming and expensive.

Thus, despite the suitability of ATR-FTIR for analysis of biologicalsamples, a significant instrumentation limitation is that an ATR-FTIRspectrometer, or an ATR attachment for an FTIR spectrometer, istypically composed of a single IRE. As the sample is placed directlyonto this IRE, this limits this technique to a single sample approachwhere the sample needs to be prepared, analysed, removed from the IRE,followed by a thorough clean of the IRE, before the next sample can beanalysed using the instrument. In the case of biofluids like bloodserum, this process is significantly elongated as there is a requirementto dry the sample, to unearth subtle biomolecular information. The timethis takes is volume dependent, but has been determined as 8 minutes for1 μL blood serum spot. As a consequence, this approach cannot beconsidered high-throughput. Reasons for this restriction include thehigh cost of current IREs, combined with engineering requirements forspecific attachments.

U.S. Pat. No. 7,255,835 (Franzen et al) discloses an apparatus andmethod for acquiring an infrared spectrum of a solubilised sample, in aFTIR microscope.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provideda sample slide for use in a spectrometer, wherein the sample slidecomprises:

a plurality of sample-receiving portions provided on a sample side ofthe slide, and

a plurality of beam-receiving portions provided on a beam-receiving sideof the slide, each beam-receiving portion being arranged opposite arespective sample-receiving portion, and wherein each beam-receivingportion is configured to act as an internal reflection element (IRE).

The provision of a sample slide having multiple sample-receivingportions allows the possibility of performing multiple measurements froma single slide without having to remove and replace the sample slidebetween successive measurements, particularly when used in combinationwith a device as described in respect of the second aspect of theinvention. This may avoid the need to remove, clean and dry a IREbetween successive measurements as is current practice, thus permittinghigh throughput ATR-FTIR analysis.

The sample-receiving portions may be aligned, e.g. may be longitudinallyaligned relative to the slide. The sample-receiving portions may defineor may be arranged as a row of sample-receiving portions. In anotherembodiment, the sample-receiving portions may be provided as or maydefine a plurality of rows of sample-receiving portions. Each row maycomprise a number of aligned sample-receiving portions.

The sample slide may comprise a number of sample-receiving portionsarranged in a row. For example, the slide may comprise foursample-receiving portions arranged in a row. This may allow for onesample-receiving portion to be used for background measurement, andthree sample-receiving portions to be used for triplicate measurementsfrom a patient. It will be appreciated that any number ofsample-receiving portions may be envisaged, depending on the number ofmeasurements wishing to be made from a single slide.

The sample slide may comprise a number of sample-receiving portionsarranged in a plurality of rows, e.g. in a grid pattern. For example,the slide may comprise 24 sample-receiving portions arranged in 4 rowsof 6 (4×6), e.g. similar to a 24-well plate arrangement. For example,the slide may comprise 96 sample-receiving portions arranged in 8 rowsof 12 (8×12), e.g. similar to a 96-well plate arrangement. This mayallow for one sample-receiving portion in each row to be used forbackground measurement, and for multiple sample-receiving portions to beused for multiple measurements from a patient, for each row. It will beappreciated that any number of sample-receiving portions may beenvisaged for such grid-like or wellplate-like arrangement, depending onthe number of measurements wishing to be made from a single slide.

Alternatively, a plurality of slides having a number of sample-receivingportions arranged in a row may be combined, e.g. may be placed adjacentto each other, e.g. laterally and/or longitudinally. This arrangementmay allow the formation of a grid pattern having a plurality ofsample-receiving portions arranged in a plurality of rows, usingmultiple “single-row” slides.

One or more sample-receiving portions may define or may consist of arecessed portion, e.g. relative to a surface of the slide, e.g. relativeto an outer surface on a sample side of the slide. Alternatively, oradditionally, one or more sample-receiving portions may be surrounded bya raised portion, e.g. relative to a surface of the slide, e.g. relativeto an outer surface on a sample side of the slide. By such provision,risk of cross-contamination between adjacent sample-receiving portionsmay be reduced or avoided.

Typically, one or more sample-receiving portions, e.g. thesample-receiving portions, may be configured to receive or support a drysample, in use. In an embodiment, one or more sample-receiving portions,e.g. the sample-receiving portions, may be configured to receive aliquid sample such as a biofluid, e.g. blood or serum, and may be driedso as to support a dry sample for use in a spectrometer.

Conveniently, the sample slide may comprise a/the internal reflectiveelement(s) IRE(s). The sample slide may be configured to act as a/theIRE(s).

The beam side of the slide may comprise a plurality of beam-receivingportions. Typically, each beam-receiving portion may be provided on thebeam side opposite a respective sample-receiving portion on the sampleside.

Each beam-receiving portion may be configured to act as an IRE.

The beam-receiving portions may be configured to permit a radiation beamto penetrate a surface of the beam-receiving portions on the beam sideof the slide. Advantageously, the beam-receiving portions may beconfigured to permit a radiation beam to penetrate a surface of abeam-receiving portion on the beam side of the slide at angle such thatthe radiation beam may be reflected on an internal surface of arespective sample-receiving portion, and may be permitted to exit theslide through the surface of the beam-receiving portion on the beamside.

The/each beam-receiving portion may comprise or may define a pluralityof grooves and/or prisms, preferably a plurality of elongate groovesand/or prisms, e.g., a plurality of aligned, parallel and/or adjacentgrooves and/or prisms.

Each groove may have or may define a first groove face and a secondgroove face. The/each first groove face may be arranged to allow aradiation beam to penetrate, e.g. inwards, a surface thereof. The/eachsecond groove face may be arranged to allow a radiation beam topenetrate, e.g. outwards, a surface thereof.

Each prism may have or may define a first prism face and a second prismface. The/each first prism face may be arranged to allow a radiationbeam to penetrate, e.g. inwards, a surface thereof. The/each secondprism face may be arranged to allow a radiation beam to penetrate, e.g.outwards, a surface thereof.

Typically, the first groove face of a groove may correspond to the firstprism face of an adjacent prism. The second groove face of a groove maycorrespond to the second prism face of an adjacent prism.

In an embodiment, the prisms may protrude outwardly, e.g. relative to asurface, e.g. a flat surface, of the slide on the beam side thereof. Inanother embodiment, the prisms may be recessed, e.g. relative to asurface, e.g. a flat surface, of the slide on the beam side thereof.Alternatively, an outer portion of the prisms may protrude outwardly,e.g. relative to a surface, e.g. a flat surface, of the slide on thebeam side thereof, and an inner portion of the prisms may be recessed,e.g. relative to a surface, e.g. a flat surface, of the slide on thebeam side thereof.

The slide may have a thickness, e.g. between a sample side and a beamside, in the range of 100-1000 μm, e.g. in the range of 200-800 μm, e.g.in the range of 300-700 μm. In some embodiments, the slide may have athickness, e.g. between a sample side and a beam side, of approximately380 μm, 525 μm or 675 μm.

The/each groove or prism may have a width, e.g. a maximum width, in therange of 50-500 μm, e.g. in the range of 50-300 μm, e.g. in the range of100-250 μm. In some embodiments, the/each groove or prism may have awidth, e.g. a maximum width, of approximately 100 μm, 150 μm, 200 μm or250 μm.

The/each groove or prism may have a depth, e.g. a maximum depth, in therange of 50-500 μm, e.g. in the range of 50-300 μm, e.g. in the range of70-200 μm. In some embodiments, the/each groove or prism may have adepth, e.g. a maximum depth, of approximately 70 μm, 100 μm, 140 μm or175 μm.

Adjacent grooves may have a spacing in the range of 0-200 μm, e.g. inthe range of 10-150 μm, e.g. in the range of 25-100 μm. In someembodiments, adjacent grooves may have a spacing of approximately 25, 50or 100 μm. When a spacing between adjacent grooves is present, anoutermost region of a respective prism comprise a levelled and/or flatportion, e.g. at a tip or outermost region thereof.

A surface, e.g. a first face and/or a second face or the/each groove orprism, may extend at an angle, e.g. relative to a surface of the slide,e.g. on a beam side thereof, in the region of 30-75°, e.g. 35-55°. Itwill be appreciated that the exact angle chosen for a given slide maydepend on the material selected for manufacture of the slide, and/or onthe expected angle of incidence of the irradiation beam. For example,the angle a groove face and/or prism face may depend on the specificmaterial used and/or on the crystalline structure thereof. When theslide is made of a <100> silicon material, a first face and/or a secondface or the/each groove or prism, may extend at an angle, e.g. relativeto a surface of the slide, e.g. on a beam side thereof, in the region of40-75°, e.g. 45-65°, e.g. approximately 55°, e.g. 54.74°. When the slideis made of a <110> silicon material, a first face and/or a second faceor the/each groove or prism, may extend at an angle, e.g. relative to asurface of the slide, e.g. on a beam side thereof, in the region of30-50°, e.g. 30-40°, e.g. approximately 35°, e.g. 35.3°.

The sample slide may be made of a material suitable for use as an IRE,for example germanium, diamond, zinc selenide, or silicon.Advantageously, the sample slide may be made of silicon. The use ofsilicon may considerably reduce the costs associated with themanufacture of the slide, and may allow the slide to be used as adisposable slide, thus avoiding the need for cleaning and drying theslide before and/or after use.

The sample slide may comprise, may be provided on or within, or may beattached to, a slide holder.

The slide holder may be configured to receive and/or hold the sampleslide.

The use of a slide holder to hold the sample slide may help prevent orreduce contact between a user and the sample slide, thus reducingcontamination associated with handling the slide. This is particularlyadvantageous when the sample slide comprises or acts as the internalreflective element(s). The use of a slide holder may also provide thesample slide with additional structural integrity, thus reducing therisk of damage or mechanical failure of the slide.

Typically, the slide holder may be made from a polymer material, e.g.ABS (acrylonitrile-butadiene-styrene), polycarbonate, polypropylene, orthe like. Alternatively, the slide holder may be made from a metallicmaterial, e.g. aluminium.

Advantageously, the slide holder may have a size corresponding to thestandard dimensions of a microscope slide. Typically, the size of theholder may be approximately 75×25×1 mm. This may help use of the sampleslide into conventional laboratories by conforming to existing handlingprocedures and avoiding the need to change common procedures.

The slide holder may be provided with a tag region suitable forreceiving an identifier, e.g. a unique identifier associated with theslide received in the holder and/or with the samples. The sampleidentifier may comprise or may be a barcode, a QR code, or the like, andmay contain information associated with the samples and/or a subject.

The sample holder may be provided with well markers which may provide auser with information regarding one or more sample-receiving regions orwells, such as one or more markers for a respective background well orrespective background wells, and one or more markers for respectivesample wells.

The sample holder may have a sample side and a beam side.

On its sample side, the slide holder may comprise or may define at leastone window or opening having a size sufficient to expose one or moresample-receiving portions. In an embodiment, the slide holder maycomprise or may define a plurality or windows or openings, each windowor opening corresponding to a respective sample-receiving portion of theslide. The windows or openings may be of a similar size to the size oftheir respective sample-receiving portions. The provision of separatewindows may provide a further physical barrier between adjacentsample-receiving portions of the slide, thus further reducing the riskof cross-contamination between adjacent sample-receiving portions. Oneor more windows or openings, e.g. each window or opening, may have asize of approximately 1-10 mm×1-10 mm. Typically, one or more windows oropenings, e.g. each window or opening, may have a size of approximately5 mm×5 mm.

On its beam side, the slide holder may comprise or may define at leastone window or opening having a size sufficient to expose thebeam-receiving portions of the slide. In an embodiment, the sampleholder may comprise or may define a window or opening on its beam sidehaving a size sufficient to expose the plurality of beam-receivingportions of the slide. In another embodiment, the sample holder maycomprise or may define a plurality or windows or openings on its beamside, each window or opening corresponding to a respectivebeam-receiving portion of the slide. The windows or openings may be of asimilar size to the size of their respective beam-receiving portions,such as approximately 1-10 mm×1-10 mm. e.g., 5 mm×5 mm.

According to a second aspect of the present invention, there is provideda device for use with a spectrometer, the device comprising:

a stage configured to receive a sample slide; and

a moving mechanism configured to move the sample slide relative to asample-measuring location of the device.

Advantageously, the sample slide may be configured to receive aplurality of samples. The sample slide may comprise a plurality ofsample-receiving portions. The provision of a moving mechanism arrangedto move the sample slide relative to the sample-measuring locationallows the analysis of multiple samples without having to remove andreplace the sample slide between successive measurements. This isadvantageous when the sample slide comprises or includes one or moreIREs or one or more IRE portions, as this avoids the need to remove,clean and dry the IRE(s) or IRE portions between successivemeasurements.

The sample slides may comprise a sample side and a beam side.

The sample side may comprise or may define a plurality ofsample-receiving portions. In an embodiment, the sample-receivingportions may be aligned, e.g. may be longitudinally aligned relative tothe slide. The sample-receiving portions may define or may be arrangedas a row of sample-receiving portions. In another embodiment, thesample-receiving portions may be provided as or may define a pluralityof rows of sample-receiving portions. Each row may comprise a number ofaligned sample-receiving portions.

The sample slide may be a sample slide according to the first aspect ofthe invention, features of which are equally applicable to the deviceaccording to a second aspect, and the features described therein arethus not repeated here merely for reasons of brevity.

The stage is configured to receive a sample slide.

The stage may be configured to receive and/or secure the sample slide.

When the sample slide is provided on or within, or is attached to, aslide holder, the stage may be configured to receive and/or secure theslide holder.

The moving mechanism may be configured to move the stage. The sampleslide and/or slide holder may be stationary relative to the stage. Insuch instance, moving the sample slide relative to the sample-measuringlocation may be caused by moving the stage.

The moving mechanism may be configured to move the sample slide. Thesample slide may be movable relative to the stage. In such instance,moving the sample slide relative to the sample-measuring location may becaused by moving the sample slide itself.

The moving mechanism may be configured to provide unidirectionalmovement. This may be particularly advantageous when using a sampleslide having a number of sample-receiving portions arranged in a row.

The moving mechanism may be configured to provide bidirectionalmovement, for example along two perpendicular axes, e.g. in a planesubstantially parallel to a surface of the slide receiving thesample(s). This may be particularly advantageous when using a sampleslide having a number of sample-receiving portions arranged in aplurality of rows, e.g. in a grid pattern.

The moving mechanism may be configured to provide or allow movement in adirection transverse, e.g. substantially perpendicular, to the slide,e.g. substantially perpendicular to an upper surface thereof. This maybe advantageous as it may allow adjustment of the position of the samplerelative to the beam without requiring adjustment of the opticalelement(s).

For example, the moving mechanism may be configured to provide or allowmovement along a longitudinal axis of the slide and/or in a directiongenerally aligned with a/the row of sample-receiving portions, and in adirection transverse, e.g. perpendicular, to the slide, e.g.substantially perpendicular to an upper surface thereof.

Alternatively, the moving mechanism may be configured to provide orallow multidirectional movement, for example along three perpendicularaxes, such as along two perpendicular axes in a plane parallel to asurface of the slide receiving the sample(s) and along a third axissubstantially perpendicular to that plane.

There may be provided at least one motor. The moving mechanism maycomprise the motor(s). The moving mechanism, e.g. motor(s), may beactuatable and/or controlled by an actuator. The actuator may becontrolled manually and/or automatically.

In use, the moving mechanism, e.g. the motor, may cause the sampleslide, slide holder and/or stage to move by a distance corresponding tothe distance between two adjacent sample-receiving portions, e.g. by adistance between a central region of two adjacent sample-receivingportions. By such provision, in use, the moving mechanism may allow asample slide to move sequentially, in order to align its samplereceiving-portions and/or beam-receiving portions with a/the radiationbeam at/in the sample-measuring location. This may allow automatedand/or high throughput measurements of multiple samples using aconventional ATR-FTIR spectrometer.

The device may further comprise at least one optical element configuredto guide a radiation beam generated by a/the spectrometer to thesample-measuring location of the device.

The at least one optical element may be provided within an opticalcompartment. The optical compartment may comprise one or more opticalelements, typically a plurality of optical elements.

One or more of the optical elements may comprise or may be one or moremirrors.

The optical elements may be arranged to guide, e.g. reflect, a radiationbeam generated by the spectrometer to a/the sample-measuring location.

The optical elements may be arranged to guide, e.g. reflect, a radiationbeam reflected by the sample slide, e.g. by an IRE thereof, to adetector of the spectrometer.

The device, e.g. optical compartment thereof, may have an inlet to allowthe radiation beam generated by the spectrometer to enter the opticalcompartment. The device, e.g. optical compartment thereof, may have anoutlet to allow the radiation beam reflected by the sample slide, e.g.by an IRE thereof, to exit the optical compartment.

The device may be configured such that, when the device is installed orfitted on a/the spectrometer, the sample measuring location of thedevice is located at or substantially at a location where a sample wouldbe placed using a conventional reflectance accessory or ATR accessoryduring use of the spectrometer. For example, the device is configuredsuch that the sample-measuring location is arranged to receive theradiation beam of the spectrometer. Thus, the device may be consideredto be an accessory for use with a conventional spectrometer.

The device, e.g. optical compartment, may be configured such that theinlet and outlet are aligned with the normal direction of the radiationbeam, i.e. with the direction of the radiation beam when the device isnot present. Thus, the device may be considered to be an accessory foruse with a conventional spectrometer.

In an embodiment, the optical compartment may have a plurality ofmirrors, e.g. three mirrors, configured to guide the radiation beam tothe IRE at a predefined angle of incidence, and return the modified beamback towards the detector. The mirrors may be flat, curved, or acombination thereof. In an embodiment, the optical compartment maycomprise 3 mirrors, e.g. flat mirrors that reflect the radiation beamfrom the inlet to the sample-measuring location, and 3 mirrors, e.g.flat mirrors, e.g. three different flat mirrors, that return theradiation beam to the outlet. In another embodiment, the opticalcompartment may comprise one or more curved mirrors, or a combination offlat mirrors and curved mirrors, and may also optionally comprise one ormore lenses or focussing elements. A person of ordinary skill in the artwill appreciate that the optical elements, e.g. mirrors, may be arrangedto guide or deliver the radiation beam to the sample-measuring locationat a desired angle, which may depend on the configuration and materialof the slide and IRE portion(s) thereof.

One or more of the optical elements may be adjustable. By suchprovision, the angle of incidence of the radiation beam on the slide maybe adjusted. This may allow the use of slides having differentconfigurations and/or of slides made from different materials.

The features described in respect of any other aspect of the inventionare equally applicable to the device according to the second aspect, andare therefore not repeated here for brevity.

According to a third aspect of the present invention, there is provideda device for use with a spectrometer, the device comprising:

at least one optical element configured to guide a radiation beamgenerated by the spectrometer to a sample-measuring location of thedevice;

a stage configured to receive a sample slide; and

a moving mechanism configured to move the sample slide relative to thesample-measuring location.

The device, e.g. optical compartment, may be configured such that theinlet and outlet are aligned with the normal direction of the radiationbeam, i.e. with the direction of the radiation beam when the device isnot present. Thus, the device may be considered to be an accessory foruse with a conventional spectrometer.

The features described in respect of any other aspect of the inventionare equally applicable to the device according to the third aspect, andare therefore not repeated here merely for reasons of brevity.

According to a fourth aspect of the present invention, there is provideda method for measuring a sample, the method comprising:

coupling a device to a spectrometer,

placing a sample slide having a plurality of sample-receiving portionson a stage of the device;

moving the sample slide relative to the sample-measuring location so asto sequentially analyse, measure or detect a plurality of samplesdisposed on the slide.

The term “coupling” will be herein understood to mean that the devicemay be used with a spectrometer. Typically, in use, the device may bephysically connected to the spectrometer, for example may be placed/laidonto a portion of the spectrometer, may be inserted within an internalportion of the spectrometer, and/or may optionally be secured orattached to the spectrometer, for example to reduce risks ofdisconnection and/or movement during analysis.

The term “analyse” will be herein understood to include measurement,detection, processing, or the like. Thus, the term “analyse” will beherein understood as referring to the measurement or detection of asample by FTIR spectrometry, and may also optionally include, but notnecessarily, further processing of the measured information, for exampleusing multivariate analysis, processing algorithms, machine learning,and/or Principal Component Analysis (PCA). For example, the use of PCAallows variables between datasets to be compared, visualised and/orhighlighted, thus identifying possible variations, e.g. biologicalvariations, between samples.

The device may have an optical compartment comprising an inlet and anoutlet, wherein the inlet is arranged to allow a radiation beamgenerated by the spectrometer to enter the optical compartment, and theoutlet is arranged to allow the radiation beam reflected by a sampleslide to exit the optical compartment.

The method may comprise guiding the radiation beam generated by thespectrometer from the inlet to the sample-measuring location of thedevice.

The method may comprise guiding the radiation beam reflected by thesample slide from the sample-measuring location to the outlet of thedevice.

The spectrometer may be an IR spectrometer, e.g. a FTIR spectrometer,typically an ATR-FTIR spectrometer, e.g. an FTIR spectrometer equippedwith or coupled to an ATR element.

The method may use Fourier transform IR (FTIR) spectroscopic analysis.In FTIR, the IR spectra may be collected in the region of 400-4000wavenumbers (cm⁻¹). Generally the IR spectra may have a resolution of 10cm⁻¹ or less, typically approximately 8 cm⁻¹ or 4 cm⁻¹. The FTIRspectroscopic analysis may employ at least 10 scans, at least 15, or atleast 30 scans. The FTIR spectroscopic analysis may employ at most 100scans, at most 50 scans, or at most 40 scans. For example, 32 scans maybe used. The scans may be co-added. As will be appreciated by theskilled person, the number of scans may be selected to optimize datacontent and data-acquisition time.

The method may use Attenuated Total Reflection (ATR)-IR spectroscopicanalysis. In some embodiments, the spectroscopic analysis may beATR-FTIR.

The method may comprise placing one or more samples on the sample slide.The method may comprise placing a sample on one or more sample-receivingportions of the slide. At least one sample, e.g. the samples, maycomprise a biological sample, e.g. a biofluid such as blood or bloodserum. Typically, when providing the sample(s) on the slide, thesample(s) may be in liquid form. At least one sample, e.g. the samples,may comprise a non-biological sample.

The method may comprise drying one or more samples, e.g. the samples.Typically, the method may comprise drying one or more samples on thesample slide. Conveniently, the method may comprise drying one or moresamples on the sample slide prior to placing the sample slide on thestage of the device. This may improve the handleability of the slide.

The method may comprise drying the sample(s) under optimised conditions.It has been discovered that, surprisingly, the drying conditions mayaffect the spectra obtained during subsequent analysis. In particular,it has been discovered that certain drying conditions may lead toimproved reproducibility of analysis and/or sharpness and/or intensityin the spectra.

The method may comprise drying the sample(s) and/or sample slide at atemperature of approximately 30-36° C., e.g., about 33-36° C., e.g.about 34.5-35.5° C., e.g. about 35° C.

The method may comprise drying the sample(s) and/or sample slide undergas flow, e.g. air flow, e.g. under controlled gas flow conditions. Theflow rate may be in the range of about 5-200 m³/h, e.g. about 10-125m³/h, e.g., about 15-115 m³/h. The flow rate may be at least 10 m³/h,e.g. at least 15 m³/h, e.g. at least 50 m³/h, e.g. at least 90 m³/h.

The method may comprise flowing a gas, e.g. air, over the sample(s), forexample for a predetermined length of time.

The method may comprise drying the sample(s) and/or sample slide suchthat the drying time of the samples and/or sample slide is approximately1-5 minutes, e.g. 1-3 minutes, e.g. approximately 2 minutes.

The features described in respect of any other aspect of the inventionare equally applicable to the method according to the fourth aspect, andare therefore not repeated here for brevity.

According to a fifth aspect of the present invention, there is provideda method for measuring a sample, the method comprising:

coupling a device to a spectrometer, the device having an opticalcompartment comprising an inlet and an outlet, wherein the inlet isarranged to allow a radiation beam generated by the spectrometer toenter the optical compartment, and the outlet is arranged to allow theradiation beam reflected by a sample slide to exit the opticalcompartment,

placing a sample slide having a plurality of sample-receiving portionson a stage of the device;

moving the sample slide relative to the sample-measuring location so asto sequentially analyse, measure or detect a plurality of samplesdisposed on the slide.

Typically, in use and/or during measurement, one or more sample, e.g.,each sample, associated with a respective sample-receiving portion, maybe dry.

The features described in respect of any other aspect of the inventionare equally applicable to the method according to the fifth aspect, andare therefore not repeated here for brevity.

According to a sixth aspect of the present invention, there is provideda method of preparing a sample for IR spectral analysis, the methodcomprising drying one or more samples on the sample slide under gas flowconditions.

It has been discovered that, surprisingly, the drying conditions mayaffect the spectra obtained during subsequent analysis. In particular,it has been discovered that certain drying conditions may lead toimproved reproducibility of analysis and/or sharpness/intensity in thespectra.

The method may comprise placing one or more samples on the sample slide.The method may comprise placing a sample on one or more sample-receivingportions of the slide. At least one sample, e.g. the samples, maycomprise a biological sample, e.g. a biofluid such as blood or bloodserum. Typically, when providing the sample(s) on the slide, thesample(s) may be in liquid form.

The method may comprise drying the samples and/or sample slide at atemperature of approximately 30-36° C., e.g., about 33-36° C., e.g.about 34.5-35.5° C., e.g. about 35° C.

The method may comprise drying the samples and/or sample slide undercontrolled gas flow conditions. The flow rate may be in the range ofabout 5-200 m³/h, e.g. about 10-125 m³/h, e.g., about 15-115 m³/h. Theflow rate may be at least 10 m³/h, e.g. at least 15 m³/h, e.g. at least50 m³/h, e.g. at least 90 m³/h.

The method may comprise flowing a gas, e.g. air, over the sample(s), forexample for a predetermined length of time.

The method may comprise drying the samples and/or sample slide such thatthe drying time of the samples and/or sample slide is approximately 1-5minutes, e.g. 1-3 minutes, e.g. approximately 2 minutes.

The features described in respect of any other aspect of the inventionare equally applicable to the method according to the sixth aspect, andare therefore not repeated here for brevity.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the invention will now be described by way of exampleonly, and with reference to the accompanying drawings, in which:

FIG. 1 is a schematic representation of the principles of ATR-IRspectroscopy;

FIG. 2 is a schematic representation of a conventional set-up showing asingle IRE for performing ATR-IR spectroscopy analysis;

FIG. 3 is a side view of a sample slide according to an embodiment ofthe present invention;

FIG. 4 is a left hand side view of the sample slide of FIG. 3 ;

FIG. 5 is a view from above of the sample slide of FIG. 3 ;

FIG. 6 is a view from below of the sample slide of FIG. 3 ;

FIG. 7 is an elevated perspective view of the sample slide of FIG. 3 ;

FIG. 8 is an elevated perspective view of an alternative embodiment of asample slide according to the present invention;

FIG. 9 is a cross-sectional view of the sample slide of FIG. 3 , showinga radiation beam reflected by the IRE;

FIG. 10 is a cross-sectional view of the sample slide of FIG. 8 ,showing a radiation beam reflected by the IRE;

FIG. 11 is an elevated perspective view showing an upper side of aholder holding the sample slide of FIG. 3 ;

FIG. 12 is an elevated perspective view showing a lower side of a holderholding the sample slide of FIG. 3 ;

FIG. 13 is an elevated perspective view showing a an upper side of aholder holding a sample slide according to another embodiment thepresent invention;

FIG. 14 is an elevated perspective view showing a lower side of theholder of FIG. 13 ;

FIG. 15 is an elevated perspective view showing a device for use with aspectrometer, according to an embodiment of the present invention;

FIG. 16 is a view from above of the device of FIG. 16 ;

FIG. 17 is a schematic cross-sectional view of an optical compartment ofthe device of FIG. 15 ;

FIG. 18A is an ATR-FTIR spectrum showing investigation of an optimumangle of incidence for an IRE in the range of 30-80°;

FIG. 18B is an ATR-FTIR spectrum showing investigation of an optimumangle of incidence for an IRE in the range of 30-50°;

FIG. 18C is an ATR-FTIR spectrum showing investigation of an optimumangle of incidence for an IRE in the range of 30-50°, following spectralpre-processing;

FIG. 19 is an ATR-FTIR spectrum showing the effect of the temperatureused to dry a sample before analysis;

FIG. 20 is an ATR-FTIR spectrum showing the effect of air flow appliedto dry a sample before analysis;

FIG. 21 is an ATR-FTIR spectrum showing the combined effect oftemperature and air flow applied to dry a sample before analysis;

FIG. 22 is a Table showing the mean area under a curve corresponding tothe peak of absorbance for the hydroxyl group for differenttemperatures;

FIG. 23 is a Table showing the mean intensity corresponding to the peakof absorbance for the amide I group for different temperatures;

FIG. 24 is a Table showing the standard deviation for measurementscorresponding to the peak of absorbance for the hydroxyl group fordifferent temperatures;

FIG. 25 is a Table showing the standard deviation for measurementscorresponding to peak of absorbance for the amide I group for differenttemperatures;

FIG. 26 is a schematic view of a method for analysing a sample accordingto an embodiment of the present invention;

FIG. 27 is an elevated perspective view showing an upper side of asample slide and holder according to another embodiment of the presentinvention;

FIG. 28 is an elevated perspective view of a front side of a device foruse with a spectrometer, according to another embodiment of the presentinvention;

FIG. 29 is an elevated perspective view of a rear side the device ofFIG. 28 ;

FIG. 30 shows a conventional Specac Quest ATR-FTIR accessory; FIG. 31shows the accessory of FIG. 30 , fitted with a device according to anembodiment of the present invention;

FIG. 32 shows the device of FIGS. 28-29 , with the sample slide andholder of FIG. 27 ;

FIG. 33 shows a comparison of spectral quality between three FTIRinstruments using a sample slide of FIG. 3 (FIGS. 33(A)-(C)), and agold-standard diamond ATR accessory (FIG. 33(D));

FIG. 34 shows alternative approaches for SIRE integration in a FTIRspectrometer and relative SNR1 values, depicting; (a) the sample slideof FIG. 3 on an adapted ATR accessory; and (b) the sample slide of FIG.3 on a specular reflectance accessory;

FIG. 35 illustrates a principal component analysis (PCA) scatter plot ofGBM (red) and non-cancer (blue) patients, with individual patientslabelled so as to observe variability between SIRE wells;

FIG. 36 is a cross-sectional view of an embodiment of a sample slideaccording to the present invention, showing certain dimensionparameters;

FIG. 37 shows the signal to noise ratio (SNR) of all 36 individual IREdesigns investigated for various combinations of width, spacing andthickness;

FIG. 38 shows a comparison of spectra obtained from designs of differentthicknesses;

FIG. 39 shows a comparison of peak intensities of IRE designs that havedifferent distances between grooves;

FIG. 40 shows a comparison between the spectra obtained from a multiwellsilicon slide according to an embodiment of the present invention, and aconventional diamond IRE;

FIG. 41 is a cross-sectional view of a specific embodiment of the sampleslide of FIG. 36 ;

FIGS. 42-44 illustrate a principal component analysis (PCA) scatter plotof three different patients undergoing treatment for melanoma; and

FIGS. 45-49 illustrate the results of a spectral analysis carried outusing a method according to an embodiment of the present invention, inrespect of bacteria samples.

DETAILED DESCRIPTION

Referring to FIG. 1 there is shown a schematic representation of theprinciples of ATR-IR spectroscopy. In FIG. 2 , these principles areillustrated in the context of the conventional set-up showing a singleIRE for performing ATR-IR spectroscopy analysis.

As shown in FIGS. 1 and 2 , “Attenuated Total Reflection” (ATR) employsan internal reflective element (IRE) 10 through which an IR beam 20 ispassed. The sample 30 is deposited directly onto the IRE 10. Thespecific refractive properties of the IRE depends on the material fromwhich the IRE is made, which can be for example diamond, germanium, zincselenide or silicon. As shown in FIG. 1 , when the IR light beam 20 ispassed through the IRE 10 at an angle θ₁ above the critical angle, thebeam 20 is internally reflected through this medium on its upper surface12 in contact with the sample 30. When the beam 20 meets the IRE andsample interface 12, this results in the production of an evanescentwave 14 which penetrates into the sample 30. The depth of thispenetration is dependent upon the refractive indices of the IRE 10 andthe sample 30, and is generally in the range of 0.5-2 μm. The beam 20,which then contains information about the sample 30, is then reflectedby the IRE 10 towards a detector.

FIGS. 3 to 6 illustrate various views of an embodiment of a sampleslide, generally denoted 100, according to an embodiment of the presentinvention.

As best shown in FIGS. 3 and 5 , in this embodiment, the slide 100 hasfour sample-receiving portions 111, 112, 113, 114 provided on a sampleside 115 of the slide 100 and arranged in a row. The provision of asample slide 100 having multiple sample-receiving portions 111-114allows the possibility of performing multiple measurements from a singleslide 100 without having to remove and replace the sample slide 100between successive measurements. This may avoid the need to remove,clean and dry an IRE between successive measurements as is currentpractice, thus permitting high throughput ATR-FTIR analysis.

In this embodiment, the surface 116 of the slide 100 on its sample side115 is substantially flat. However, in other embodiments, one or more ofthe sample-receiving portions 111-114 may define or may consist of arecessed portion, e.g. relative to an upper surface 116 of the slide100. Alternatively, or additionally, one or more of the sample-receivingportions 111-114 may be surrounded by a raised portion, e.g. relative toan upper surface 116 of the slide 100.

As best shown in FIG. 6 , the sample slide 100 also has fourbeam-receiving portions 121,122,123,124 provided on a beam side 125 ofthe slide 100. Each beam-receiving portion 121-124 is arranged oppositea respective sample-receiving portion 111-114. Advantageously, eachbeam-receiving portion 121-124 is configured to act as an internalreflection element (IRE). For example, in this embodiment, onesample-receiving portion 121 may be used for background measurement, andthree sample-receiving portions 122,123,124 to be used for triplicatemeasurements of a subject's sample, or for measurements of threeseparate samples from one or more subjects.

Advantageously, the slide 100 contains and acts as the internalreflective elements (IRE(s)) required to perform ATR-FTIR analysis.

As best shown in FIGS. 4 and 6 , the beam-receiving portions 121-124 areconfigured to permit a radiation beam to penetrate a surface of thebeam-receiving portions 121-124 on the beam side 125 of the slide 100.

Each beam-receiving portion 121-124 defines has a plurality of elongategrooves 126 and prisms 127. Conveniently, each beam-receiving portion121-124 defines has a plurality of aligned, parallel and adjacentgrooves 126 and prisms 127.

As best shown in FIGS. 4, 9 and 10 , each groove 126 has a first grooveface 128 and a second groove face 129. Each prism 127 also has a firstprism face 128, corresponding to the first groove face, and a secondprism face 129, corresponding to the second groove face 129.

Each first groove face 128 or first prism face 128 is arranged to allowa radiation beam 20 to penetrate inwards a surface of a respective prism127. Each second groove face 129 or second prism face 129 is arranged toallow a radiation beam 20 to penetrate outwards a surface of arespective prism 127.

In the embodiment of FIGS. 7 and 9 , the prisms 127 protrude outwardlyrelative to a lower surface 117 of the slide 100 on the beam side 125thereof.

In the embodiment of FIGS. 8 and 10 , the prisms 127 are recessedrelative to a lower surface 117 of the slide 100 on the beam side 125thereof.

Alternative embodiments may be envisaged in which an outer portion ofthe prisms 127 may protrude outwardly relative to a lower surface 117 ofthe slide 100 on the beam side 125 thereof, and an inner portion of theprisms 127 may be recessed relative to the lower surface 117.

In the embodiments described herein, the thickness (t) of the slide 100was manufactured to test a number of configurations and dimensions, andeach type of slide was tested in the following thicknesses: 380 μm, 525μm and 675 μm.

Various dimensions of grooves 126 and prisms 127 were envisaged andexperimented with, as illustrated in Table 1 below:

TABLE 1 width (μm) depth (μm) spacing (μm) Design No. 100 70.6 25 1 50 2100 3 150 105.9 25 4 50 5 100 6 200 141.2 25 7 50 8 100 9 250 176.5 2510 50 11 100 12

Further investigation regarding the configuration and design of thebeam-receiving portions 121-124 acting as an IRE element for slide 100,was carried out, as represented by FIGS. 36-41 .

As shown in FIG. 36 , each beam-receiving portions 121-124 acting as IREelements has a thickness (t), and grooves 126 have a width (w) and areseparated by spacing (s). Testing was performed for width (w) of 100 μm,150 μm, 200 μm and 250 μm, spacing (s) of 25 μm, 50 μm and 100 μm, andthickness (t) of 380 μm, 525 μm and 675 μm. All slides were made ofsilicon.

It was expected that a greater groove width (w) may allow more IR lightto couple into the IRE elements (beam-receiving portions 121-124) andtherefore improve signal throughput and intensity. It was also thoughtthat decreasing the spacing between adjacent grooves (s) may reducelight scattering below the IRE well, reducing noise arising from thestray light recombining at the detector. Lastly, signal quality wasexpected to improve as the IRE thickness (t) decreased. This is becausethe effective path length of the IR beam through the IRE (beam-receivingportions 121-124) is reduced, thus preventing specific IR energy bandsbeing absorbed into the material of the IRE itself which would result ina loss of signal at specific wavenumbers.

SNR was calculated by taking the average signal value, x, of the Amide Iband region (1625 cm⁻¹ to 1675 cm⁻¹) and dividing this by the standarddeviation, σ, found across a region of the spectra where a high amountof noise can be found (1825 cm⁻¹ to 1875 cm⁻¹). Equation 1 expressesthis:SNR= x _(signal)/σ_(noise)  Equation (1)

The results indicating signal-to-noise ratio (SNR) of all 36 individualIRE designs (representing each combination of width, spacing andthickness) are shown in FIG. 37 . Colours indicate the 3 thicknessesinvestigated (red=380 μm, green=525 μm and blue=675 μm).

It was concluded from FIG. 37 that a thickness of 380 μm would generatespectra with a more desirable SNR. This was an unexpected outcome asalthough the thickness of the IRE is expected to link to a higherinformation content below 1500 wavenumbers the significant improvementin signal-to-noise ratio demonstrates the superiority of the 380 micronapproach.

A comparison of spectra obtained from designs of different thicknessesis shown in FIG. 38 . For this experiment, spacing (s) between grooves,and groove width (w) were kept constant in each case

It can be se from FIG. 38 that the peaks of the 380 μm thick IREs arehigher, indicating higher spectral intensity. It can also be seen thatan improved signal intensity can be observed at the amide 1 and 2 bandregions in thinner IREs. The improvement of thinner silicon IREs overthicker elements is further affirmed by way of a one-way ANOVA and Tukeypost hoc comparison which confirms a significant difference existsbetween spectra obtained from 380 μm thick IREs and spectra obtainedfrom 525 μm and 675 μm thick IREs (p<0.001 for a 95% confidenceinterval). Statistical analysis was carried out using Minitab.

A comparison of peak intensities of IRE designs that have differentdistances between v-grooves is shown in FIG. 39 (green=25 μm, blue=50 μmand red=100 μm).

This did not suggest a significant impact of groove width (w) andspacing (s) on SNR. There did not appear to be a discerniblerelationship between SNR and either groove width (w) or spacing (s)between grooves. However, when looking at the intensity of spectra whereparameters width and spacing are kept constant, it appeared that asmaller spacing between grooves resulted in greater signal intensity(FIG. 39 ). This observation was consistent at all t and w values.

Silicon naturally absorbs light of certain infrared frequency ranges.More specifically, silicon can cut-off signal below 1500 cm⁻¹wavenumbers as the beam is allowed to travel through the silicon forlong enough. The effects of this can be reduced by decreasing thedistance the beam travels through the silicon crystal. FIG. 40 shows acomparison between the spectra obtained from a multiwell silicon IRE ofthe present invention, and a conventional diamond IRE obtained fromSpecac Ltd.

It can be seen from FIG. 40 that signals below 1500 cm⁻¹ can still beacquired using the silicon IREs. However, effects of silicon latticeabsorption can still be observed. At about 610 cm⁻¹ wavenumbers a troughcan be seen in the spectra acquired using Silicon IREs. This is to beexpected, and is indicative of signal being lost as a result of internallattice vibrations.

The use of silicon as the material used to manufacture the slide 100 isparticularly advantageous as this considerably reduces the costsassociated with the manufacture of the slide 100, and allows the slide100 to be used and marketed as a disposable slide, thus avoiding theneed for cleaning and drying the slide before and/or after use.

Based on the above observations, an advantageous embodiment of a slideaccording to the present invention was fabricated, illustrated in FIG.41 , having four 5×5 mm beam-receiving portions 121-124, and having a380 μm thickness. The grooves were 250 μm wide, with a 25 μm spacingbetween them.

Additionally, optimum angles for the first face 128 and second face 129or the grooves 126 and prisms 127 were investigated, as explained inmore detail with reference to FIG. 18 . A suitable angle for a slide 100made of silicon was found to be about 54.74° for a <100> silicon slide,and about 35.3° for a <110> silicon slide. It will be appreciated thatthe exact angle chosen for a give slide may depend on the materialselected for manufacture of the slide, and/or on the expected angle ofincidence of the irradiation beam.

Referring now to FIGS. 9 and 10 there are shown cross-sectional views ofthe sample slide 100 of FIGS. 7 and 8 , respectively, showing aradiation beam 20 being reflected by the IRE structure of the slide 100.It will be understood that the path of the beam is shown forillustration purposes, and that the actual beam path may depart from thepath shown in FIGS. 9 and 10 . For example, without wishing to be boundby theory, it is thought that, after the beam 20 has entered the slide100, for example through a surface of first face 128 of a prism 127, thebeam 20 may travel along a direction of the prisms 127, before beingreflected on an internal surface 116 of the slide 100 and exiting theslide 100, for example through a second face 129 of another prism 127.

Referring to FIGS. 11 and 12 there are shown an upper side and anunderside of a holder 130 configured for holding the sample slide 100 ofFIG. 3 .

The use of a slide holder 130 to hold the sample slide 100 may helpprevent or reduce contact between a user and the sample slide 100, thusreducing contamination associated with handling the slide 100, which isparticularly advantageous as the sample slide 100 comprises and acts asthe internal reflective element(s). The use of a slide holder 130 alsoprovides the sample slide with additional structural integrity, thusreducing the risk of damage or mechanical failure or the slide 100.

Advantageously, the slide holder 130 has a size corresponding to thestandard dimensions of a microscope slide, typically approximately75×25×1 mm. This may help use of the sample slide 100 into conventionallaboratories by conforming to existing handling procedures and avoidingthe need to change common procedures.

The sample holder has a sample side 136 and a beam side 137.

On its sample side 136, the slide holder 130 has four windows 131-134,each window corresponding to and being of a similar size to a respectivesample-receiving portion 111-114 of the slide 100. Typically, eachwindow 131-134 and sample-receiving portion 111-114 has a size ofapproximately 5 mm×5 mm, which may allow each sample-receiving portion111-114 to hold approximately 1-10 μL of sample, in use.

On its beam side 137, the slide holder 130 has a window 135 having asize sufficient to expose the beam-receiving portions 121-124 of theslide 100. In an alternative embodiment, it may be envisaged that theside holder 130 may comprise or may define a plurality or windows on itsbeam side 137, each window or opening corresponding to a respectivebeam-receiving portion 121-124 of the slide 100.

The slide holder 130 is provided with a tag region 138 suitable forreceiving an identifier 139, which may be associated with the slide 100received in the holder 130 and with the samples deposited on the slide100.

Another embodiment of a holder, denoted 130 b, is shown in FIG. 27 . Theholder 130 b of FIG. 27 is generally similar to the holder 130 of FIG.11 , like parts being denoted by like numerals, supplemented by thesuffix “b”. The holder 130 b of FIG. 27 has an arrow A showing thedirection of movement of the holder 130 b and slide 100 b, in use, toalign, sequentially, each of the beam-receiving portions (not shown)located opposite their respective sample-receiving portions 111 b-114 bwith a beam of a spectrometer. The holder 130 b also has markings0,1,2,3 to allow easy labelling and referencing of each sample-receivingportion 111 b-114 b.

Referring now to FIGS. 13 and 14 there are shown an upper side and anunderside, respectively, of a holder 230 and corresponding sample slide200, according to another embodiment the present invention. In thisembodiment, the slide 200 has a 96-well plate configuration, that is,has 96 samples receiving portions 211 arranged in 8 rows of 12. Eachwell-receiving portion 211 has a corresponding beam-receiving portion221 on a beam side 227 of the slide 200.

On its sample side 236, the slide holder 230 has 96 windows 231, eachwindow corresponding to and being of a similar size to a respectivesample-receiving portion 211 of the slide 200.

On its beam side 237, the slide holder 230 has a window 235 having asize sufficient to expose the beam-receiving portions 221 of the slide200. In an alternative embodiment, it may be envisaged that the slideholder 230 may comprise or may define a plurality or windows on its beamside 237, each window or opening corresponding to a respectivebeam-receiving portion 221 of the slide 200.

It will be appreciated that any number of sample-receiving portions maybe envisaged for such grid-like or well-plate-like arrangement,depending on the number of measurements wishing to be made from a singleslide 200.

FIGS. 15 and 16 show an elevated perspective view and a top view,respectively, of a device, generally denoted 300, for use with aspectrometer, according to an embodiment of the present invention.

The device 300 comprises an optical compartment 310 which has aplurality of optical elements 311-316 configured to guide a radiationbeam 20 generated by the spectrometer to a sample-measuring location 320(shown in FIG. 17 ) of the device 300.

The device also has a stage 330 configured to receive a sample slide340. In this embodiment, the sample slide 340 is a slide 100 asdescribed with reference to FIGS. 3 to 12 , and is provided within aholder 350 which is similar to the holder 130 described with referenceto FIGS. 11 and 12 . Thus, in this embodiment, the stage 330 isconfigured to receive and secure the slide holder 350 which holds thesample slide 340.

The device 300 contains a moving mechanism 360 which is configured tomove the sample slide 340 relative to the sample-measuring location 320.In this embodiment, since the sample slide 340 is provided within aslide holder 350, the moving mechanism 360 is configured to move theslide holder 350 which holds the sample slide 340, relative to thesample-measuring location 320.

Because the slide 340 has four sample-receiving portions 341-344, theprovision of a moving mechanism 360 arranged to move the sample slide340 relative to the sample-measuring location 320 allows the analysis ofmultiple samples without having to remove and replace the sample slide340 between successive measurements. This is advantageous when thesample slide comprises or includes one or more IREs, as this avoids theneed to remove, clean and dry the IRE(s) between successivemeasurements.

In this embodiment, the moving mechanism 360 is configured to move thestage 330. Since the sample slide 340 and slide holder 350 arestationary relative to the stage 330, moving the stage 330 causes thesample slide 340 to be moved relative to the sample-measuring location320.

In this embodiment, because the sample slide 340 has foursample-receiving portions 341-344 aligned in a longitudinal direction,the moving mechanism 360 is configured to provide unidirectionalmovement in the direction of alignment of the sample-receiving portions341-344.

However, if using a different slide, for example a slide 200 asdescribed with reference to FIGS. 13 and 14 , the moving mechanism 360may be configured to provide bidirectional movement, for example alongtwo perpendicular axes, in order to allow each of the 96sample-receiving portions 211 to be sequentially aligned with thesample-measuring location 320.

The moving mechanism 360 comprises a motor 362 for moving the stage 330.The moving mechanism 360, e.g. motor 362, can be controlled by anactuator (not shown), which can be activated manually and/orautomatically.

In use, the moving mechanism 360, e.g. motor 362, causes the stage 330,and therefore the sample slide 340, to move by a distance correspondingto the distance between two adjacent sample-receiving portions 341-344.By such provision, in use, the moving mechanism 360 allow the sampleslide 340 to move sequentially, in order to align its samplereceiving-portions 341-344 and beam-receiving portions with theradiation beam 20 in the sample-measuring location 320. This may allowautomated and/or high throughput measurements of multiple samples usinga conventional ATR-FTIR spectrometer.

An embodiment of the optical compartment 310 of the device 300 is bestshown in FIG. 17 .

The optical compartment 310 has a plurality of optical elements 311-316configured to guide a radiation beam 20 generated by the spectrometer toa sample-measuring location 320 of the device 300. In this embodiment,the optical elements 311-316 are mirrors.

The optical compartment 310 has walls that define an optical chamber317. The optical compartment has an inlet 318 provided in a wallthereof, to allow the radiation beam 20 generated by the spectrometer toenter the optical compartment 310 and the optical camber thereof. Theoptical compartment has an outlet 319 provided in a wall thereof, e.g. awall opposite the wall containing the inlet 318, to allow the radiationbeam 20 reflected by the sample slide 340, to exit the opticalcompartment 310. The optical compartment 310 also has an opening (notshown) in an upper wall thereof to allow the reflected beam 20 to hitthe sample slide 340 at the sample-measuring location 320.

Typically, the inlet 318 and the outlet 319 are aligned with the normaldirection of the radiation beam 20, i.e. aligned with the direction ofthe radiation beam 20 when the device 300 is not present. Thus, thedevice 300 may be considered to be accessory for use with a conventionalspectrometer.

Optical elements 311-313 are arranged to guide the radiation beam 20 tothe sample-measuring location 320 at a predefined angle of incidence,and optical elements 314-316 are arranged to return the modified beamback towards the outlet 319. A person of ordinary skill in the art willappreciate that the optical elements 311-316, e.g. mirrors, may bearranged to guide or deliver the radiation beam 20 to thesample-measuring location 320 at a desired angle, which may depend onthe configuration and material of the slide and IRE portion(s) thereof.

Without wishing to be bound by theory, it is thought that an adequateangle of incidence for the radiation beam may be similar to or may be inthe region of the angle of the the/a face 128,129 of the slide 100. Forexample in an embodiment with a <110> silicon slide having a face128,129 angle of about 35.3°, the angle of incidence may be adjusted tobe approximately 32°.

One or more of the optical elements 311-316, e.g. each optical element311-316, may be adjustable. By such provision, the angle of incidence ofthe radiation beam on the slide may be adjusted. This may allow the useof slides having different configurations and/or of slides made fromdifferent materials.

FIGS. 28-29 show an elevated perspective view, from front and rear,respectively, of a device, generally denoted 300 b, for use with aspectrometer, according to another embodiment of the present invention.The device 300 b is generally similar to the device 300 of FIGS. 15-16 ,like parts being denoted by like numerals, but supplemented by thesuffix “b”. However, in the embodiment of FIGS. 28-29 , the device 300 bdoes not have an optical compartment.

The device 300 b has a stage 330 b configured to receive a sample slide340 b. The device 300 b also has a moving mechanism 360 b which isconfigured to move the sample slide 340 b relative to a sample-measuringlocation 320 b. In this embodiment, since the sample slide 340 b isprovided within a slide holder 350 b, the moving mechanism 360 b isconfigured to move the slide holder 350 b which holds the sample slide340 b, relative to the sample-measuring location 320 b. The device 300 bhas a switch 375 b, and control buttons 371 b-374 b to control movementof the stage 330 b and/or align a desired sample receiving-portions 341b-344 b and beam-receiving portions with a radiation beam (not shown) atthe sample-measuring location 320 b.

A view of the device 300 b with a sample slide 100 b and holder 130 b isshown in FIG. 32 . In this embodiment, the sample slide and holder aresimilar to the slide 100 b and holder 130 b as described with referenceto FIG. 27 .

The stage 330 b also has openings 331 b-334 b, each opening beingconfigured to be substantially adjacent or aligned with a respectivesample receiving-portion 341 b-344 b and beam-receiving portion of thesample slide 340 b.

It will be appreciated that, similarly to device 300 of FIGS. 15-16 ,device 300 b may be configured for bi-directional movement of a sampleslide with a plurality of wells arranged in rows, or movement of aplurality of sample slides in holders 100 b and 130 b.

FIG. 30 shows a conventional Specac Quest ATR accessory 500 comprisingan optical body 501 and a lid 502 which has a stage 503 to receive asample on a conventional IRE and a compression arm 504 to maintain theIRE in position during analysis.

FIG. 31 shows the accessory 500′ of FIG. 30 , with its lid 502 removed,and replaced by the device 300 b of FIG. 28-29 . The device 300 b isthus sized and configured to be connected to and fitted onto thespectrometer optical body 501. By such provision, the device 300 b isconfigured such that, when the device 300 b is installed or fitted onthe spectrometer body 501, the sample measuring location of the device300 b is located at or substantially at a location where a sample wouldbe placed using a conventional sample slide during use of the accessory500′. Thus, the device 300 b may be considered to be an accessory foruse with a conventional spectrometer.

A person of skill in the art will appreciate that other embodiments maybe made which are sized and configured to fit other types ofconventional spectrometers, such as, but not limited to as examples aVeemax optical accessory or those of a Perkin Elmer Spectrum Two orThermo Fisher iS5 or Agilent Cary series, while using the samecombination of a stage configured to receive a sample slide, and amoving mechanism configured to move the sample slide relative to asample-measuring location.

With reference to the FTIR spectra shown in FIGS. 18 to 21 , thesespectra contain two peaks of absorption that are of particular interest:the strong peak of absorption in the 3200-3500 cm⁻¹ region which ischaracteristic of a water OH group, and the strong peak of absorptionaround 1690 cm⁻¹ which is characteristic of a primary amide group (asfound in proteins).

Investigation of optimum angles of incidence are described withreference to FIGS. 18A-18C.

FIG. 18A is an ATR-FTIR spectrum showing absorbance for different anglesof incidence in the range of 30-80°. In FIG. 18B, the angles ofincidence have been reduced to a range of 30-50°, as these angles wereidentified as providing optimum results. FIG. 18C is similar to thespectrum of FIG. 18B, but following spectral pre-processing, focussingon the absorbance region characteristic of primary amides. Spectralpre-processing typically involves a wavenumber selection approach toeffectively ‘cut’ a fingerprint region of the spectrum, where typicallythe majority of biological molecules will present. Spectralpre-processing also typically includes a baseline correction(rubberband), to adjust for any scattering properties in the spectrum.Finally, and vector normalisation can be applied, which reduces theinfluence of sample inconsistencies, such as differences in thickness.

In the spectra shown in FIGS. 19 to 21 , protein-containing serumsamples were analysed, under varying drying conditions.

It has been discovered that, surprisingly, the drying conditions mayaffect the quality and reproducibility of the spectra obtained duringsubsequent analysis. In particular, it has been discovered that certaindrying conditions may lead to improved reproducibility of analysisand/or sharpness in the spectra.

FIG. 19 is an ATR-FTIR spectrum showing the effect of the temperatureused to dry a sample before analysis. A 1 μL serum sample was depositedon a sample slide of the present invention and dried for 8 minutes attemperatures of T1=25° C., T2=30° C., and T3=35° C. It can be seen thatdrying the sample at 35° C. not only reduced the water content in thesample (smaller absorbance in the 3200-3500 cm⁻¹ region), but alsoimproved the absorption reading in the sample in relation to the primaryamide group (absorption around 1690 cm⁻¹), compared to samples dried forthe same duration at lower temperatures.

FIG. 20 is an ATR-FTIR spectrum showing the effect of air flow appliedto dry a sample before analysis. A 1 μL serum sample was deposited on asample slide of the present invention and dried for 8 minutes at roomtemperature of approximately 20° C. under air flows corresponding to afan voltage of V1=5V, V2=12V, and V3=14V. It was measured that a fanvoltage of 5V corresponded to a flow rate of approximately 15 m³/h, afan voltage of 12V corresponded to a flow rate of approximately 99 m³/h,and a fan voltage of 14V corresponded to a flow rate of approximately113 m³/h. It can be seen that drying the sample using a 14V fan voltage(hence under higher air flow) not only reduced the water content in thesample (smaller absorbance in the 3200-3500 cm⁻¹ region), but alsoimproved the absorption reading in the sample in relation to the primaryamide group (absorption around 1690 cm⁻¹), compared to samples dried forthe same duration at lower fan voltages (hence under lower air flow).

FIG. 21 is an ATR-FTIR spectrum showing the combined effect oftemperature and air flow applied to dry a sample before analysis. A 1 μLserum sample was deposited on a sample slide of the present inventionand dried for 8 minutes at 35° C. under air flows corresponding to a fanvoltage of V1=5V, and V3=14V. It can be seen that, while a sample driedat 35° C. under low air flow (fan voltage of 5V) showed low moisturecontent and good primary amide absorbance peak, increasing the flow rate(fan voltage of 14V) further reduced the water content in the sample,and improved the sharpness of the primary amide peak of absorption.

FIG. 22 is a Table showing the mean area (in arbitrary absorbance units²(au²)) under a curve corresponding to the peak of absorbance for thehydroxyl group (absorbance in the 3200-3500 cm⁻¹ region) for differenttemperatures and drying times. It is clear from FIG. 22 that increasingthe drying temperature to 35° C. reduced the water content in the samplecompared to samples dried at lower temperatures. It can also be seenthat increasing fan voltage (and hence the air flow rate) also reducedthe water content in the sample compared to samples dried at lower fanvoltages. Finally, it can be seen that the combined effect of a dryingtemperature of 35° C. with a high air flow (12V or 14V fan voltages)produced the best results. In particular, the drying time required todry the sample was significantly reduced at 35° C. compared to lowertemperatures. For example, a value of less than 12 was achieved after adrying time of 2 minutes under a temperature of 35° C. and with fanvoltage of 5V, 12V or 14V, whereas, at 30° C., the water content washigher even after 6 minutes drying time.

FIG. 23 is a Table showing the mean intensity corresponding to the peakof absorbance for the amide I group for different temperatures. It canbe observed that increasing the temperature and air flow each led toincreased absorbance intensity characteristic of the amide I group. Inother words, there was a clear correlation between the dryness of thesample (low water content as observed in FIG. 22 ) and the measuredintensity of the amide I peak absorbance in the sample, which wasunexpected.

FIG. 24 is a Table showing the standard deviation for measurementscorresponding to the peak of absorbance for the hydroxyl group fordifferent temperatures. FIG. 25 is a Table showing the standarddeviation for measurements corresponding to peak of absorbance for theamide I group for different temperatures.

The standard deviation is representative of the reproducibility, with alower standard deviation meaning better reproducibility. It can be seenfrom FIGS. 24 and 25 that drying the sample at higher temperature (35°C.) and under air flow each improved reproducibility of the ATR-FTIRanalysis, with the best results being achieved for a combination of adrying temperature of 35° C. with application of air flow.

FIG. 26 is a schematic view of a method 400 for analysing a sampleaccording to an embodiment of the present invention.

Step 410 illustrates the step of collecting a serum sample 415. Wholeblood was drawn from a patient. Following this the blood underwent acentrifugation step to isolate the blood serum other blood components.Typically, a minimum of 5 ml of serum is required per patient. Sampleswere snap-frozen for storage and thawed to room temperature foranalysis.

Step 420 illustrates the step of dispensing a serum sample 415 on onto asample slide 430. The sample slide 430 was a slide according to anembodiment of the present invention as described with reference to FIGS.3-7 and 11-12 .

5 μL of a samples were pipetted into wells 432, 433 and 434, while well431 was left empty, to be used as background control.

Step 440 illustrates the step of drying a batch of slides 430 a, 430 b,430 c. The slides were stacked and placed in a drying unit 442 fordrying. The drying unit 442 was set to a temperature of about 35° C. andthe slides were allowed to dry for about 2 minutes using a 5V fan toprovide an air flow rate of about 15 m³/h.

Step 450 illustrates the step of performing spectral analysis of thesamples.

The instrument used in the analysis of the samples is a Spectrum 2™ FTIRspectrometer from Perkin Elmer. This spectrometer is fitted with anaccessory device 300 as described in the embodiment of the presentinvention referring to FIGS. 15-17 . This facilitates high-throughputanalysis of samples. The slide 430 was provided with an identifier 439fixed on one end of the slide 430 to easily and reliably identify theoriginal of the samples. In this embodiment, the closest well 431 to theidentifier 439 was used as a ‘background’ well 431. Typically, theslides 430 were placed on the stage 330 of the device 300 such that thebackground well 431 is analysed first by the spectrometer. A person ofskill in the art will appreciate that the purpose of a blank well is toserve as a background scan of the environment for the spectrometerinstrument. This collects all spectral information from the environmentand removes it from the data collected from the subsequent serumsamples. Therefore, a background measurement is typically carried outbefore analysis of samples to be analysed. This ensures that, in thecontext of analysing serum samples, important information from the serumis not obscured by components in the surrounding environment.

As described with reference to FIGS. 15-17 , once ATR-FTIR measurementis complete for first well 431, the apparatus 300 moves the slide 430relative to the sample-measuring location 320, for the sample in thesecond well 432 to be analysed. Thus, each sample in wells431,432,433,434 is analysed by ATR-FTIR spectrometry without the need toremove the slide 430 between measurements.

The spectrometer was configured in the following manner: a resolution of4 cm⁻¹, a 4 cm⁻¹ aperture, and with 32 scans per sample and background.This is a standard ATR-FTIR spectrometer setting which allows spectra tobe taken typically in under a minute.

Step 460 illustrates the step of processing the data and presenting theinformation to a user via a user interface 462. The present methodallows results to be delivered in real time and presented to the user aswith a simple interface 462 displaying the result with the percentagelevel of confidence.

Once analysis has been completed, the slide 430, which also acts as theIRE for each well 431,432,433,434, may be disposed of appropriately, aswill typically be treated as biological waste for disposal.Alternatively, the slide 430 may be stored for future reference, orcleaned and re-used as appropriate to the application.

Experimental Data

Use of Sample Slide with Conventional Spectrometers (FIG. 33 )

A sample slide according to an embodiment of the present invention wasprepared, consistent with the embodiment of FIGS. 3-6 . The slide wasmade of silicon.

A sample was prepared with human pooled serum, and applied to each ofthe three sample wells, the first well being used for baselinereference. The same sample slide was used throughout so as to reduce anydifferences arising from sample preparation. A single spectrum wasobtained from each sample well and recorded. Spectra were thenpre-processed using a rubberband baseline correction followed by vectornormalisation. These steps aim to negate any background effects arisingfrom unwanted light scattering, as well as sample variations such asthickness which can have multiplicative effects on the spectra.Pre-processing thus makes all spectra comparable.

To compare data regarding spectral quality, and thus system performance,values of signal-to-noise (SNR) were extracted. A higher SNR can beregarded as preferable as important spectral information is larger incomparison to the unwanted background noise. SNR can be calculated bycomparing intensity values of a ‘signal’ region against a ‘noise’region. The amide I peak of a biological spectrum is often the mostintense due to fundamental vibrations from protein and thus the maximumabsorbance value of this peak is often used as a signal region. Noisevalues can be obtained from anywhere in the IR spectrum that is free ofvibrational modes found in biological samples and modes arising fromambient conditions. The regions between 4000-3700 cm⁻¹, 2800-2500 cm⁻¹,2000-1800 cm⁻¹ and below 900 cm⁻¹ are commonly chosen noise regions. Inthis instance, maximum absorbance values between 1900-1850 cm⁻¹ and900-850 cm⁻¹ were selected as two separate measures of signal quality.The former area was chosen to avoid contributions from the waterovertone region; the latter, to encompass loss of sensitivity in lowerwavenumbers due to the use of silicon, whilst also addressinglimitations in detector sensitivity.

FIG. 33 shows a comparison of spectral quality between three FTIRinstruments using a sample slide of FIG. 3 (FIGS. 33(A)-(C)), and agold-standard diamond ATR accessory (FIG. 33(D), and in particular:

-   -   (A) Perkin Elmer Spectrum 2 FTIR,    -   (B) Thermo Fisher Nicolet is 5 FTIR,    -   (C) Agilent Technologies Cary 670 FTIR, and    -   (D) Perkin Elmer Spectrum 2 with uATR accessory (gold standard).

SNR values are given for each set of spectra, with the amide I peakabsorbance compare to the maximum absorbance value in the 1900-1850 cm⁻¹region (SNR1), and the maximum absorbance in the 900-850 cm⁻¹ region(SNR2)

It can be seen from FIG. 33(D) that the gold standard approach of usinga commercially available ATR accessory with integrated diamond crystalyields high quality spectra with high SNR. FIGS. 33(A)-(C) show thatusing the samples slides according to an embodiment of the presentinvention, integrated onto a universal ATR accessory (using a SlideIndexing unit), in conjunction with three other commercially availablespectrometers, the Perkin Elmer Spectrum 2 FTIR spectrometer producesspectra with equivalent quality to the gold standard approach (FIG.33(A)). Good quality spectra are also acquired from Thermo FisherNicolet iS 5 (FIG. 33(B)) and Agilent Cary 670 (FIG. 33(C)) systems.

Comparison of Silicon IREs (‘SIREs’) (FIG. 34 )

Alternative approaches to the approach described in relation to FIG. 33were explored using adapted IRE interfaces with commercially availableATR (FIG. 34(a)) and specular reflectance accessories (FIG. 34(b)). Asshown in FIG. 34 , the SNR for each approach is distinctly lower thanthe spectral quality obtained from a gold-standard approach such as adiamond IRE system, and considerably less than the system describedabove and investigated in relation to FIG. 33 .

Clinical Classification Study on SIREs (FIG. 35 )

The diagnostic performance of ATR-FTIR has been established usingproof-of-concept studies using diamond IRE based ATR. Theseretrospective studies determined that brain tumour patients could bedistinguished at sensitivities and specificities of 92.8% and 91.5%respectively. In order to investigate the diagnostic performance of aSIRE-based approach, a small classification study was conducted on 15glioblastoma multiforme (GBM) and 15 control patients. A small studysuch as this would provide an indication of potential performance ofthis new approach. One important consideration with a small dataset isthat some computational methods would not be suitable, due to the riskof overfitting and insufficient validation.

For all patients, 3 μL of serum was pipetted onto each of the wells ‘1’,‘2’ and ‘3’ on a sample slide according to the embodiment of FIG. 3 ,and allowed to dry at room temperature (20-22° C.). Three spectra wereobtained per well resulting in 9 spectra per patient, and an overalltotal of 270 spectra. Spectra were pre-processed by cutting to thefingerprint region (900-1000 cm⁻¹), second-order differentiated andvector normalised. As the number of spectra is less than the number ofvariables in the dataset, supervised analysis was deemed unsuitable andas such multivariate analysis, in this case principal component analysis(PCA), was conducted to observe differences between cancer andnon-cancer patients (FIG. 35 ).

PCA is a technique used to emphasize variation and highlight patterns ina dataset. In the present case, the PCA transformation was carried outin 2 dimensions, with the first principal component (PC1) beingassociated with the wavenumber and absorbance showing the largestvariance in the dataset, and the second principal component (PC2) beingassociated with the wavenumber and absorbance showing the second largestvariance in the dataset. In other words, PCA analysis reduces a spectruminto variables that account for variance within the dataset. As such,when these variables are compared against each other in a scatterplot,separation between classes in the axes suggests biological variation.Conversely, relative closeness or overlap suggests biologicalsimilarity.

If cancer and non-cancer appear to separate in a PCA scatter plot, thissuggest that the two classes are distinguishable using the FTIRapproach. What can be seen in FIG. 35 is that there is a distinct splitbetween cancer and non-cancer patients, with very little overlap. Thisis promising for any subsequent analysis, such as the use ofclassification algorithms that would extract sensitivity andspecificity, as an unsupervised approach is able to initially identifyspectral differences indicative of disease status.

A further clinical classification study was carried out for patientssuffering from melanoma. The approach taken was similar to the abovestudy as illustrated in FIG. 35 , but with regard melanoma-type cancersrather than brain tumors.

FIGS. 42-44 shows the results of a principal component analysis (PCA)carried out for three different patients being monitored during thecourse of their treatment. The sample analysis in each case was carriedout as described in the method 400 of FIG. 26 , followed by PCAanalysis.

With reference to FIG. 42 , samples taken from five different patientvisits were analysed. The patient had melanoma in visit 1, and arelapsed occurred at visit 3. As shown in FIG. 42 , PCA analysis of theresults showed that the samples associated with visit 2 weredistinctively different and separated from the other samples. Theresults shown on FIG. 42 clearly show that the present method allowidentification of the presence or absence of cancer (in this casemelanoma) in a patient's biological sample.

Similarly, with reference to FIG. 43 , the patient had melanoma in visit1, and a relapsed occurred at visit 3. Again, PCA analysis of theresults showed that the samples associated with visit 2 (no melanoma)were distinctively different and separated from the other samples(melanoma).

The patient associated with FIG. 44 was different in the sense that thepatient had no melanoma in visits 1, 3 and 4, but a relapsed occurred atvisit 2. Thus, in this case, the samples which were distinctivelydifferent and separated from the other samples were the samples whichidentified the presence melanoma.

FIGS. 45-49 illustrate the results of spectral analyses carried outusing a method according to an embodiment of the present invention, inrespect of bacteria samples.

FIG. 45 shows average spectral data highlighting spectral differences inthe six different bacteria families that the 86 samples that wereanalysed belong to.

FIG. 46 shows average spectral data highlighting spectral differences inbacteria according to bacteria genus.

FIGS. 47-48 show PCA analysis PC1 vs PC2, and PC1 vs PC3, respectively,highlighting the differences between bacteria families.

FIG. 49 illustrates the PC loadings spectral discrimination associatedwith the PCA analysis of FIGS. 47-48 . Separation between bacterialfamilies is shown via scatter plots and corresponding loadings show thatthis is due to distinctive spectral regions.

Thus, it can be see that the methods and systems of the presentinvention are not limited to the investigation of cancerous samples, butmay also be applied to numerous other applications, including forexample the investigation and identification of different types ofbacteria families of genus. Further, it will be appreciated that themethods and systems of the present invention are not limited to theinvestigation of biological samples, but may also be applied tomeasuring or testing non-biological applications, for whichspectrometry, e.g. ATR_FTIR spectrometry, may lead to useful resultsinterpretation and/or classification.

It will be appreciated that the described embodiments are not meant tolimit the scope of the present invention, and the present invention maybe implemented using variations of the described examples.

The invention claimed is:
 1. A slide, comprising: a) a ample-receivingportion provided on a sample side of a slide; b) a beam-receivingportion provided on a beam side of said slide, said beam-receivingportion being arranged opposite said sample-receiving portion, whereinsaid beam-receiving portion comprises an internal reflection element(IRE); and c) a thickness between said beam-receiving portions and saidopposite sample-receiving portion selected from the group consisting of380 μm and 675 μm.
 2. The slide according to claim 1, wherein saidsample-receiving portion comprises a recessed portion surrounded by araised portion.
 3. The slide according to claim 1, wherein saidsample-receiving portion is configured to receive or support a drysample.
 4. The slide according to claim 1, wherein said internalreflection element comprises adjacent grooves that are aligned orparallel and adjacent prisms that are aligned or parallel.
 5. The slideaccording to claim 4, wherein each of said adjacent groove has a widthin the range of 50-500 μm.
 6. The slide according to claim 1, whereineach of said adjacent groove are spaced apart in the range of 0-200 μm.7. The slide according to claim 1, wherein said slide further comprisessilicon.
 8. The slide according to claim 1, further comprising a slideholder, wherein said slide is on, within, or attached to said slideholder.
 9. The slide of claim 1, wherein said slide is configured tointerface with a FTIR spectrometer.
 10. The slide of claim 1, whereinsaid slide is configured to interface with an ATR-FTIR spectrometer. 11.The slide of claim 1, wherein said internal reflection element comprisesan infra-red transmissible material.
 12. The slide of claim 11, whereinsaid infra-red transmissible material is selected from the groupconsisting of diamond, germanium, zinc selenide and silicon.
 13. Amethod of preparing a sample for IR spectral analysis, the methodcomprising: drying one or more samples on a slide at a temperature ofapproximately 30-36° C. and/or under a gas flow rate of at least 50m³/h, wherein said slide comprises: a) a sample-receiving portionprovided on a sample side of said slide; b) a beam-receiving portionprovided on a beam side of said slide, said beam-receiving portion,wherein said beam-receiving portion comprises an internal reflectionelement (IRE), and c) a thickness between said beam-receiving portionand said opposite sample-receiving portion selected from the groupconsisting of 380 μm and 675 μm.
 14. The method of claim 13, whereinsaid temperature ranges between 34.5 to 35.5° C.
 15. The method of claim13, wherein said gas flow rate is at least 90 m³/h.
 16. The method ofclaim 13, wherein said internal reflection element comprises aninfra-red transmissible material.
 17. The method of claim 16, whereinsaid infra-red transmissible material is selected from the groupconsisting of diamond, germanium, zinc selenide and silicon.